Flt3 Is Required for MLL-ENL-Induced Leukemogenesis In Vivo, but Not for LSC Function in Vitro

Abstract 2450

Flt3 is a type III tyrosine kinase receptor expressed on hematopoietic multipotential progenitors and more downstream progenitor cells in the mouse bone marrow (BM).Flt3 is one of the most frequently mutated genes in acute myeloblastic leukemia (AML) patients, leading to constitutive activation. Several Flt3 kinase inhibitors are in clinical development in AML with Flt3 mutations, although disappointing results suggest that Flt3-inhibitors as monotherapy are unlikely to become reality. Wild type (wt) Flt3, which differs in its signaling characteristics and downstream targets from mutated Flt3, is highly expressed in particular in AML and ALL (acute lymphoblastic leukemia) with MLL (Mixed Lineage Leukemia) rearrangements. These observations suggest a role for wt Flt3 in pathogenesis as well, although little experimental or clinical evidence has been published yet supporting this notion. To our knowledge, there is only one report suggesting that wt Flt3 is required for the development of leukemia in a murine model of c-cbl mutation-induced leukemogenesis (Rathinam C, et al. Cancer Cell 2010). For this reason, we focused on the role of wt Flt3 in leukemogenesis and using leukemia mouse models established by retroviral transduction of MLL-ENL, MLL-AF9 and AML1-ETO9a. Mice transplanted with MLL-ENL-transduced Flt3−/− BM cells showed a higher survival rate (wt 61.3%, n=34 vs Flt3−/− 96.8%, n=31, median follow-up period; 255 days, p=0.038). No difference in survival was observed in the MLL-AF9 and AML1-ETO9a models however. Surprisingly, MLL-ENL-transduced Flt3−/− BM cells showed no significant difference in serial replating capacity of BM cells in methylcellulose colony forming assays in vitro compared to wt BM cells, and putative lin-kit+ leukemic stem cells (LSCs) did not express Flt3. In vivo, however, putative MLL-ENL LSCs, but not MLL-AF9 LSCs expressed Flt3. Furthermore, in non- and pre-leukemic MLL-ENL-transduced mice BM cells the frequency of lin-kit+ putative LSCs among MLL-ENL positive populations was decreased in Flt3−/− group (wt 1.468% vs Flt3−/− 0.466%, p=0.031). Flt3+ lin-kit+ populations were increased in MLL-ENL transduced leukemic wt mice compared to non-leukemic mice (leukemic mice 26.7% vs non-leukemic mice 9.9%, p=0.028). These results suggest that Flt3 signaling is required for MLL-ENL-induced leukemogenesis and maintenance of LSCs in vivo, but not in vitro. Importantly, the data suggest that the in vitro replatable leukemia stem cell (LSC) paradigm might not reflect LSC function or phenotype in vivo.